Runx2, a gene expressed in an osteoblast-specific fashion, is a transcriptional factor responsible for the determination of differentiation from undifferentiated mesenchymal cells into the osteoblast series and the maturity of chondrocytes. The expression of Runx2 in osteoblasts is high in immature stages and decreases with the maturity of osteoblasts. It has been reported that Runx2 promotes osteoblast differentiation in the initial stage and suppresses late-phase differentiation and final differentiation into osteocytes (non-patent documents 1 and 2). Runx2 is also expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes, possessing both the action of promoting the differentiation and maturity of immature chondrocytes and the action of inhibiting the formation of permanent chondrocytes (non-patent documents 3-5). Also, an analysis using animals with a modified Runx2 gene is ongoing.
Runx2 occurs in an isoform starting with exon 1 (type II Runx2) and another isoform starting with exon 2 (type I Runx2), which undergo transcriptional regulation by a distal promoter and a proximal promoter, respectively (non-patent document 6). Both isoforms are expressed in osteoblasts and chondrocytes (non-patent document 3). Thereof, absolutely no report is available on the proximal promoter; with regard to the distal promoter, however, a transcriptional regulatory region 1.5 kb upstream of the exon 1 has been reported using cultured cells (non-patent document 7). Also available is a report of transgenic mice generated using a transcriptional regulatory region 3 kb upstream of the exon 1, but they exhibit an expression pattern totally different from the physiological expression pattern for Runx2 (non-patent document 8).
There are 2.3 kb and 3.2 kb type I collagen promoters that have been used so far as promoters (DNAs) to allow expression by osteoblasts. With the 3.2 kb type I collagen promoter, expression induction is possible from the early stage of osteoblast differentiation; the expression is highly induced in dental odontoblasts, tendons, and fascia, and weakly induced in subcutaneous fibroblasts. The 2.3 kb type I collagen promoter is unable to induce the expression in the early stage of osteoblast differentiation. The same also strongly induces the expression in dental odontoblasts as well as in osteoblasts, and weakly induces the expression in subcutaneous fibroblasts. Also, the expression induction potential in cultured cells is extremely low. Besides, a 1.3 kb osteocalcin promoter is available, but this is not so commonly used for induction of gene expression in osteoblasts because the expression induction level in living organisms is rather low. Also, the expression thereof is restricted to mature osteoblasts. Therefore, it has been impossible to express a gene comprising a coding region in time of need in a required amount in an osteoblast-specific fashion.